Abstract:

There is provided an emulsion composition for therapeutic administration
comprising: (a) at least one mono-(electron transfer agent) phosphate
derivative; (b) at least one di-electron transfer agent phosphate
derivative; wherein the amount of transfer agent phosphate derivatives is
no less than equimolar to the amount of di-electron transfer agent
phosphate; and (c) a suitable carrier.

7. The composition of claim 1, wherein the mono-(electron transfer agent)
phosphate compound is a mono-.alpha.-tocopheryl monophosphate, and the
di-(electron transfer agent) phosphate compound is a
di-.alpha.-tocopheryl monophosphate.

9. The composition of claim 8, wherein the molar ratio of the
mono-(electron transfer agent) phosphate compound to the di-(electron
transfer agent) phosphate compound is in the range of 85:15 to 65:35.

10. The composition of claim 9, wherein the molar ratio of the
mono-(electron transfer agent) phosphate compound to the di-(electron
transfer agent) phosphate compound is about 70:30.

20. The method of claim 14, wherein the mono-(electron transfer agent)
phosphate compound is a mono-.alpha.-tocopheryl monophosphate, and the
di-(electron transfer agent) phosphate compound is a
di-.alpha.-tocopheryl monophosphate.

25. The method of claim 24, wherein the complexing agent is lauryl-imino
di-propionic acid or arginine.

26. A method of increasing the surface activity and detergency of a
composition; the method comprising adding a surface active agent selected
from a mono-tocopheryl phosphate, a salt of mono-tocopheryl phosphate,
and a mixture thereof to the composition.

27. The method of claim 26, wherein the surface active agent is mono
α-tocopheryl monophosphate, a salt of mono α-tocopheryl
monophosphate, or a mixture thereof.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001]This application is a continuation of U.S. patent application Ser.
No. 10/416775, which is a National Stage filing of International
Application No. PCT/AU01/01475, filed Nov. 14, 2001, which claims the
benefit of Australian Patent Application No. PR5499, filed Jun. 6, 2001,
and U.S. Provisional Application No. 60/247997, filed Nov. 14, 2000, all
of which are incorporated herein by reference in their entirety.

[0004]In this specification, where a document, act or item of knowledge is
referred to or discussed, this reference or discussion is not to be taken
as an admission that the document, act or item of knowledge or any
combination thereof was at the priority date:

[0005](a) part of common general knowledge; or

[0006](b) known to be relevant to an attempt to solve any problem with
which this specification is concerned.

[0007]Whilst the following discussion concerns tocopherol and dermal
therapy, it is also to be understood that the same principles apply to
any application in which a therapeutic formulation containing electron
transfer agents may be used.

[0008]The skin is the largest organ of the body, and, among other things,
functions to protect the internal organs from external chemical, physical
and pathological hazards. Normal skin is composed of an outer epidermis
covering sub dermal layers, where each layer comprises different
sections. The outer cornified layer of the epidermis possesses properties
of strength, flexibility, high electrical impedance and dryness that
retards penetration and proliferation of microorganisms. The cornified
protective layer is formed by the migration of maturing keratinocytes
that are formed at the junction of the dermis and epidermis.

[0009]Vitamin E (tocopherol) is an essential part of skin dynamics and is
known to be very important for skin health, with deficiency manifesting
as a cornified, scaly delicate skin, thickened epidermis, scaling,
lesions, chronic infection, inflammation and erythema. Vitamin E is the
main naturally occurring lipid soluble agent protecting the skin from
stress, and is the main lipid soluble agent protecting the cell membrane
lipids from peroxidation.

[0010]Skin is subject to constant stress due to exposure to everyday
elements--sun, wind and water. As a result, it is common for many topical
personal care products such as lotions, moisturizers, shampoo and
conditioners to contain vitamin E in various forms to assist in
maintaining skin health. In order to assist in maintaining skin health,
it is necessary for the vitamin E to reach the target area of the dermis.
The most direct method of achieving this targeting is to apply a topical
formulation to the affected area. However, topical application of vitamin
E to the skin using current formulations has variable success due to the
skin's ability to erect an impenetrable barrier to many outside elements.
It is critical to provide for the penetration of vitamin E through the
epidermis to the dermis.

[0011]The use of free tocopherol is avoided because it is unstable,
therefore suitable derivatives must be found. In the alimentary canal, it
has been found that there is lipase activity which releases free
tocopherol from the esters of tocopherol, typically the acetate ester.
This lipase activity enables the use of tocopheryl acetate as a
nutritional source of Vitamin E.

[0012]In contrast, the surface of the skin is deficient in lipase activity
unless it is infected with microorganisms that are able to digest
sebaceous excretions. Thus tocopheryl acetate must first diffuse through
the epidermis into the vital derma, where the cells have a very limited
lipase activity which releases the Vitamin E. It is believed that topical
formulations using tocopherol acetate have not been able to deliver
adequate tocopherol beyond the epidermal layers, and therefore provide
little benefit. Since tocopheryl acetate is a lipidic material requiring
formulation with an oil in water emulsion, absorption from such a
formulation is less than optimal.

[0013]The epidermis is permeable to water soluble substances, such as
tocopheryl phosphate. Until now producers of formulations containing
tocopheryl phosphate utilized monotocopheryl phosphate isolated from the
mixture produced during phosphorylation. The phosphorylation has been
typically achieved using phosphorous oxychloride. The product was
purified because it was believed that the by-products were deleterious to
the efficacy of the monotocopheryl phosphate because not all the
by-products were water soluble. The perceived deleterious effects were
considered significant enough to justify the cost of complicated
purification processes. Typically, the purification is performed by using
ethanol to extract the ditocopheryl phosphate and free tocopherol
by-products.

SUMMARY OF THE INVENTION

[0014]It has been found that the use of a non-purified or semi-purified
electron transfer agent phosphorylation therapeutic product is
efficacious. In particular, the non-water soluble di-(electron transfer
agent) phosphate derivatives do not have a deleterious effect on the
efficacy of the therapeutic product and may even provide a synergistic
effect which results in beneficial properties which enhance the dermal
penetration and/or efficacy of the mono-(electron transfer agent)
phosphate derivatives.

[0015]According to a first aspect of the invention, there is provided a
therapeutic emulsion composition comprising the following:

[0018]wherein the amount of mono-(electron transfer agent) phosphate
derivative is no less than equimolar to the amount of di-(electron
transfer agent) phosphate derivative; and

[0019](c) a suitable carrier.

[0020]According to a second aspect of the invention, there is provided a
method for administering to a subject electron transfer agent phosphate
derivatives comprising the step of administering a therapeutic emulsion
composition comprising the following:

[0023]wherein the amount of mono-(electron transfer agent) phosphate
derivative is no less than equimolar to the amount of di-(electron
transfer agent) phosphate derivative; and

[0024](c) a suitable carrier.

[0025]The term "electron transfer agent" is used herein to refer to the
class of chemicals which may be phosphorylated and which (in the
non-phosphorylated form) can accept an electron to generate a relatively
stable molecular radical or accept two electrons to allow the compound to
participate in a reversible redox system. Examples of classes of electron
transfer agent compounds that may be phosphorylated include hydroxy
chromans including alpha, beta and gamma tocopherol, tocols and
tocotrienols in enantiomeric and racemic forms; quinols being the reduced
forms of vitamin K1 and ubiquinone; hydroxy carotenoids including
retinol; and ascorbic acid.

[0026]The phosphate derivatives of electron transfer agents comprise
compounds covalently bound by means of an oxygen to the phosphorus atom
of a phosphate group. The oxygen atom is typically derived from a
hydroxyl group on the electron transfer agents. The phosphate derivative
may exist in the form of a free phosphate acid, a salt thereof, a
di-phosphate ester thereby including two molecules of electron transfer
agent, a mixed ester including two different compounds selected from
electron transfer agents, a phosphatidyl compound wherein the free
phosphate oxygen forms a bond with an alkyl or substituted alkyl group,
or a complex with a complexing agent selected from amphoteric surfactant,
cationic surfactant, amino acids having nitrogen functional groups or
proteins rich in these ammo acids.

[0027]Examples of acceptable salts of mono-tocopherol phosphate
derivatives are selected from the group consisting of the di-sodium,
di-potassium, di-lithium, di magnesium, mono-sodium, mono-potassium,
mono-lithium, or mono-magnesium salts or mixtures thereof. Preferably,
the acceptable salts of di-tocopheryl phosphate derivatives are selected
from the sodium, potassium, lithium or magnesium salts. The di-tocopheryl
phosphate derivatives will usually only form a salt in the environment
required to form the di-metal salts of mono-tocopheryl phosphate
derivatives.

[0028]Preferably, the molar ratio of mono-(electron transfer agent)
phosphate derivatives to di-(electron transfer agent) phosphate
derivatives is in the range from 85:15 to 65:35. There must be enough
di-(electron transfer agent) phosphate derivatives to form an emulsion
and prevent the mono-(electron transfer agent) phosphate derivatives from
going completely into solution, but not so much di-(electron transfer
agent) phosphate derivatives that there is precipitation.

[0029]The mixture of mono-(electron transfer agent) phosphate derivatives
and di-(electron transfer agent) phosphate derivatives can be prepared by
recombining the purified individual components or by using the unpurified
or semi-purified reaction product of a phosphorylation process.
Preferably, the mixture is obtained by using the reaction product of a
phosphorylation process. The source of a mixture of tocopheryl phosphate
derivatives is preferably the reaction product of the phosphorylation of
tocopherol using P4O10.

[0030]The term "acceptable carrier" is used herein to refer to a carrier
considered by those skilled in the drug, food or cosmetic arts to be
non-toxic when used to treat humans, animals or plant in parenteral or
enteral formulations. The carrier chosen will depend on the route of
administration. Ingestible formulations includes tablets, capsules,
powders, chewable tablets, capsules, oral suspensions, children's
formulations, enteral feeds, nutraceuticals and functional foods. For a
topical application, the carrier typically comprises hydrophilic
substances such as water, glycerol, polyethyleneglycol, sorbitol or
propanol. For example, the composition could be used as a shampoo, hair
conditioner, moisturizing cream or lotion or lipstick as a topical
application.

[0031]According to a third aspect of the invention, there is a process for
preparing a therapeutic emulsion composition containing phosphate
derivatives of electron transfer agents comprising the steps of:

[0032](a) phosphorylating one or more electron transfer agents using
P4O10 to form a mixture of at least one mono-(electron transfer
agent) phosphate derivative and at least one di-(electron transfer agent)
phosphate derivative;

[0033]wherein the amount of mono-(electron transfer agent) phosphate
derivative is no less than equimolar to the amount of di-(electron
transfer agent) phosphate derivative; and

[0035]The mono-(electron transfer agent) phosphate derivatives have good
water solubility, therefore before they can be absorbed into the skin or
hair an aqueous topically applied composition must dry. In contrast,
di-(electron transfer agent) phosphate derivatives are not water soluble
and cause the formation of an unstable emulsion when emulsified with
water and other hydrophilic solvents. Without wishing to be bound by
theory, it is noted that skin is hydrophobic so when the composition is
spread onto the skin, the droplets in the emulsion are attracted to the
skin. The micelles become unstable near a hydrophobic surface and break
so the mono-(electron transfer agent) phosphate derivatives are released
onto the skin. The mono-(electron transfer agent) phosphate derivatives
can then diffuse through the epidermis into the derma. Therefore,
di-(electron transfer agent) phosphate derivatives (once considered a
nuisance by-product) function as an effective spreading agent for the
mono-(electron transfer agent) phosphate derivatives.

[0036]Again, without wishing to be bound by theory, it is considered
necessary for a product which is being ingested to have several types of
surface activity including detergency and appropriate surface tension to
facilitate absorption. Mono-(electron transfer agent) phosphate
derivatives may have strong detergency but do not have sufficient surface
tension effects. Therefore, the mixture of mono-(electron transfer agent)
phosphate derivatives and di-(electron transfer agent) phosphate
derivatives having self-emulsification properties which include both
types of surface activity, that is, strong detergency and strong surface
tensions, will be better absorbed, especially in the small intestine.

[0037]It has surprisingly further been found that pure mono-tocopheryl
phosphate and its salts are powerful surface active agents and detergents
giving a stable foam.

[0038]According to a fourth aspect of the invention, there is provided a
detergent composition comprising a surface active agent selected from the
group consisting of mono-tocopheryl phosphate, its salts and mixtures
thereof.

[0039]There is also provided a method of increasing the surface activity
and detergency of a composition by adding a surface active agent selected
from the group consisting of mono-tocopheryl phosphate, its salts and
mixtures thereof.

[0040]Again, whilst not wishing to be bound by theory, it is thought that
this detergent property may be due to the fact that mono-tocopheryl
phosphate is in the form of a polar head and a non-polar tail. In
contrast, di-tocopheryl phosphate has 2 non-polar tails and a polar
central group which makes it surface active but it is not a detergent
because at high concentrations it accumulates in the surface layer of the
composition and acts as a foam breaker because the surface becomes
predominantly non-polar.

EXAMPLES

[0041]The invention will now be further illustrated and explained by
reference to the following non-limiting examples.

Example 1

[0042]In this example, a therapeutic formulation according to the
invention was prepared using tocopherol as the electron transfer agent.

Preparation of the Tocopheryl Phosphate Mixture

[0043]Take 500 g dl-alpha-tocopherol and mix with a high shear mixer 4
aliquots each of 21 g of P4O10 at 12 minute intervals, holding
the temperature above 60° C. While the mixture is still hot, add
over 1.5 hours 91.5 g of sodium hydroxide which has been dissolved in
62.5 g of water at 50° C. to hydrolyze and neutralize the
tocopheryl phosphates. The product was cooled to ambient temperature then
further cooled with liquid nitrogen to give a brittle product that was
ground to a powder and dried under vacuum.

[0045]The dried powder was dispersed in water as a 5% solution. 10 ml of
this solution was applied to the hands to give a satisfactory application
of the tocopheryl phosphates to the skin.

Example 2

[0046]The skin penetration properties of a mixture of mono- and
di-tocopheryl phosphates according to the invention were compared to
tocopheryl acetate.

Test Formulations

[0047]The test materials are made up on the basis of 5% mixed actives
(mono-tocopheryl phosphate (TP), di-tocopheryl phosphate (T2P) or
tocopheryl acetate) in a vehicle consisting of 95/5 distilled
water/ethanol with pH adjusted (if necessary to 6.5-7.0 with citric acid
or dilute NaOH).

TP and T2P (Mixed Sodium Salts)

[0048]A slurry of 6.25 w/w % of 80% mixed TP and T2P in 93.75 w/w % of the
95/5 water/ethanol mixture was prepared.

[0051]The test formulations are evaluated in vitro human skin penetration
studies. Samples are analyzed for the mono- and di-tocopheryl phosphates,
free alpha-tocopherol, and tocopheryl acetate by high performance liquid
chromatography (HPLC). The tests ate conducted by DermTech International
(San Diego, Calif.). Human cadaver skin samples are obtained and
prepared. Each formulation is evaluated on triplicate sections from each
donor at a topically applied dose of 5 μL/cm2. Receptor solutions
are collected over 48 hours at pre-selected time intervals. After 48
hours the skin surface is washed with isopropyl alcohol, and the skin is
collected and split into epidermis and dermis. The skin sections are
extracted with isopropyl alcohol. All collected samples are processed and
assayed for tocopherol, tocopheryl acetate, tocopheryl phosphate and
di-tocopheryl phosphate.

[0052]Mass balance from the samples is between 80-120% of the applied
dose.

[0053]No tocopherols are observed in the receptor solution. This could be
a result of amounts being below limits of detection, or degradation of
the various tocopherol species into other, as yet uncharacterized,
compounds.

[0054]The results demonstrate that the inclusion of 20 to 30% of T2P in
the formulation did not have a deleterious effect on the performance of
the tocopheryl phosphate product. Further, both of the TP/T2P mixtures
were more efficiently transported into the dermis than the tocopheryl
acetate product

Example 3

[0055]In this example, a mixture was prepared comprising mono-ubiquinyl
phosphate and di-ubiquinyl phosphate made according to the invention.

[0056]100 g of ubiquinone was partially dissolved in 200 ml of hot glacial
acetic acid. To the vigorously stirred solution, small amounts of zinc
(total of 30 g) were added until the solution changed from yellow to
green and then became colorless. The hot solution was filtered and the
unreacted zinc was washed 2 more times (50 ml) with hot glacial acetic
acid to recover any remaining ubiquinol. Glacial acetic acid was removed
from the ubiquinol by vacuum distillation or by cooling the solution to
0° C. and filtering off the crystallized ubiquinol. To further
remove any traces of acetic acid, the ubiquinol was placed under high
vacuum (1 mm) Hg) for a period of 2 hours.

[0057]The ubiquinol product was treated immediately by heating to
100° C. and adding 33 g of P4O10. The mixture was
stirred for 3 hours and then 500 mm water was introduced slowly into the
mixture. The temperature of the reaction was maintained just below
boiling point for a further 1 hour. Removal of water yielded ubiquinyl
phosphates and phosphoric acid. The phosphoric acid was partially removed
by further washes with hot water.

[0058]The final product consisted of 139 g of mono-ubiquinyl phosphate,
di-ubiquinyl phosphate and phosphoric acid. The product was analyzed by
31PNMR and the molar ratio of mono-ubiquinyl phosphate: di-ubiquinyl
phosphate was 76:24.

Example 4

[0059]In this example, the surface active properties of mono-tocopheryl
phosphate was investigated. 0.1 g of pure di-sodium mono-tocopheryl
phosphate was dissolved in 10 ml of pure distilled water in a 50 ml
cylindrical stoppered vessel. The vessel was shaken on a test tube
agitator and the headspace filled with stable foam. The foam was examined
on a daily basis and showed complete stability for one day and then
slowly degraded over the rest of the four-day period.